Heart failure (HF), the leading cause of death for both men and women in the US, is ultimately precipitated by a loss of functional cardiomyocytes from the heart following cardiac injury, and currently the only therapeutic strategy that addresses cardiomyocyte loss is heart transplantation. Unfortunately, the demand for donor hearts far outweighs the supply and, thus, the field of cardiac regeneration, which aims to repair dysfunctional myocardium following injury, holds great promise for the alleviation HF-associated morbidity and mortality. Indeed, several recent preclinical studies have now reported improved cardiac function following stem cell (SC) treatment in large animal models of cardiac injury as well as patients suffering from cardiac dysfunction as a consequence of myocardial infarction (MI); however, the mechanism underlying the improved cardiac function still remains unclear. Given that the contractile performance of severely ischemic hearts was significantly improved by engrafting SCs into the injured hearts, and the essential role that cardiac myofilaments play in signal transduction and reception, we hypothesize that cellular signaling between SCs and at-risk cardiomyocytes within the peri-infarct border zone (BZ), results in the stimulation of signaling pathways involved in the alteration of myofilament protein post-translational modifications, and that these changes can improve contractility and reduce stress-induced cardiac cell death. Herein, we are undertaking a systems approach to delineate the role of myofilaments in SC-associated cardiac regeneration utilizing top-down mass spectrometry (MS)-based proteomics in combination with in vitro and in vivo functional studies. The specific aims of this proposal include: 1) Identify myofilament PTM changes associated with SC treatment via a novel top-down proteomics strategy and correlate the observed changes with alterations in myocyte contractility by performing mechanical measurements of skinned myocardial preparations isolated from control pig left ventricle and from the peri-infarct BZ of MI and stem cell-treated MI pigs. 2) Identify the sites of PKA and PKC phosphorylation in myofilaments from the peri-infarct BZ of MI and stem cell-treated MI pig myocardium using top-down proteomics and determine the effects of these modifications on cardiac contractility using skinned myocardial preparations. 3) Assess how cardiac regeneration-associated PTMs influence cardiac contractility by exchanging recombinant myofilament proteins, engineered to express site-specific PTMs, with endogenous myofilament proteins in skinned fiber bundles and performing mechanical measurements. The success of the proposed research will offer new insights into the molecular mechanisms underlying SC-associated cardiac regeneration and may facilitate the development of improved cell-based therapies, which could aid in the battle against HF development post-MI.